The purpose of this research is to examine the mechanism by which cooperativity is induced in the inhibition of 5'-nucleotidase by Concanavalin A (Con A). Recent studies from our laboratory have shown that the coperativity in mammary ascites tumor cells is sensitive to drugs which perturb the cellular cytoskeleton and to intracellular Ca ions. Effects of the cytoskeletal perturbants can be prevented by agents which raise the concentration of cellular cyclic AMP. We propose a series of experiments to investigate the mechanism of the cooperativity change and relate it to physiological processes which occur in other cell types. The ascites cells, fibroblasts, lymphocytes, isolated rat mammary membranes and bovine milk fat globule membranes will be used in these investigations. In intact cells treated with cytoskeletal perturbant drugs (e.g. colchicine, cytochalasins) the relationship between the cooperativity change and changes in specific cytoskeletal elements (e.g. microtubules, microfilaments) will be examined by nonionic detergent extractions, electron microscopy and electrophoresis. Since a change in the association of the nucleotidase with the membrane is proposed to be important, a study of the nucleotidase-membrane interaction will be performed. Proteolysis, crosslinking, extraction, electrophoresis and enzyme activity measurements will be used to try to relate cooperativity and kinetics changes to specific membrane proteins. Reconstitution of the nucleotidase into liposomes of defined lipid composition with or without other Con A receptors will be performed to define further the components and conditions important to the cooperativity. Antibody specific to 5' -nucleotidase will be isolated and used in additional perturbation and labeling studies to define the mode of association of the enzyme with the membrane. Other cell types, which show responses to more physiological perturbations, will be examined to see if they exhibit a change in cooperativity resulting from physiological responses.